Skip to main content
. Author manuscript; available in PMC: 2024 Mar 1.
Published in final edited form as: Nat Methods. 2023 Jul 20;20(9):1426–1436. doi: 10.1038/s41592-023-01959-z

Figure 1. In vitro properties of nLightG and nLightR.

Figure 1

a, Structural models of nLightG (left) and nLightR (right) generated using ColabFold39. b, Representative images of HEK293T cells and neurons expressing nLightG or nLightR before/after application of NE (10 μM) and corresponding pixel-wise ΔF/F0 heatmaps. White insets indicate surface expression of the indicators. Scale bars, 10 μm (HEK293T), 20 μm (neurons). c, Left, timelapse of fluorescence of response (ΔF/F0) of nLightG in HEK293T (dark green) or neurons (light green) upon application of NE (10 μM) followed by application of trazodone (Trz, 10 μM). Right, quantification of maximal ΔF/F0 responses from left. Two-tailed Students t-test with Welch’s correction. n = 3 independent experiments with n=22 cells (HEK293T), n=18 cells (neurons). P = 2.979×10-16 (HEK293T), P = 8.558×10-17 (neurons). d, Same as in c for nLightR. n = 3 independent experiments with n = 21 cells (HEK293T), n=6 cells (neurons). P = 5.922×10-32 (HEK293T), P = 0.004 (neurons). e, Fluorescence dose-response curves of nLightG (left) and GRABNE1m (right) for NE and DA in HEK293T cells normalized to the maximum ΔF/F0 for NE for each indicator. Datapoints were fitted with four-parameter dose-response curves to determine EC50 values. n=6,5,3 wells for nLightG with NE, nLightG with DA, and GRABNE with NE/DA, respectively. f, Same as in e for nLightR with n=3 wells. All data are shown as mean ± SEM. g, Representative heatmap of nLightG fluorescence response (ΔF/F0 %) in an outside-out membrane patch from nLightG-expressing HEK293T cells (n = 6 independent experiments). Scale bar, 50 μm. h, ON and OFF kinetics of nLightG (top) and GRABNE1m (bottom) after ultrafast (<0.5 ms) switching of the perfusion pipette. Dots represent the average of 5 applications. Kinetic parameters were obtained using single-exponential fits on the average of all trials. i, Statistical comparison of kinetic parameters. Unpaired two-tailed Student’s t test. n=6 and 5 patches for nLightG and GRABNE1m, respectively. P = 3.0 ×10-8 (τON), P = 2.7 ×10-5 (τOFF). Mean ± standard deviation are shown. j, Five consecutive NE applications on nLightG (5 μM, 1 s). Images were acquired at 100 Hz. All experiments were repeated at least three times with similar results.